JBSNF-000088   中文名称: 6-甲氧基-3-吡啶羧胺

human NNMT (IC50 = 1.8 µM); monkey NNMT (IC50 = 2.8 µM); mouse NNMT (IC50 = 5.0 µM)

JBSNF-000088（6-甲酰胺烟酰胺）针对 U2OS 或分泌型 3T3L1 细胞的 IC50 值分别为 1.6 μM 和 6.3 μM [1]。

JBSNF-000088（6-甲酰胺烟酰胺）（50 毫克/公斤；4 周粉末功效）在第 21 天表现出统计显着的体重减轻百分比，并导致糖尿病血糖显着降低 [1] JBSNF -000088（50 毫克/公斤；4 周粉末功效）临界强饲法；每天两次，持续 4 周）导致终点耐受性显着改善，并在第 28 天使终点耐受性正常化 [1]。 JBSNF-000088（1 mg/kg；静脉拓扑；持续时间 4 小时）在三个重复周期中产生 21 mL/min·kg 和 0.7 L/kg 的低组织清除率，静脉注射后半衰期非常短（ 0.5 )[1 JBSNF-000088（10 mg/kg；灌胃；4 小时持续时间）导致 Cmax 为 3568 ng/mL，Tmax 值为 0.5 小时，表明快速腔内吸收和破坏，半衰期为0.4小时灌胃。

基于荧光的NNMT酶分析[1]
用荧光酶法测定NNMT活性。NNMT反应中形成的MNA在KOH和甲酸的存在下与苯乙酮反应，形成荧光产物2,7-萘啶JBSNF-000088使用人、小鼠和猴子NNMT酶进行筛选。不同浓度的抑制剂与酶在室温下预孵育30分钟。通过在37°C下加入SAM和烟酰胺混合物（分别用于人、小鼠和猴子NNMT测定的7µM、20µM、8µM SAM和6µM、二十µM、9µM烟酰胺）60分钟来引发反应。最终的测定反应混合物包含100mM Tris-Hcl pH 7.5的缓冲液、0.04%BSA、2mM二硫苏糖醇和1%DMSO。在孵育结束时，通过加入乙醇：苯乙酮混合物（75%乙醇：25%苯乙酮）和用50%乙醇制备的5M氢氧化钾来停止反应。将反应物温育15分钟，然后加入100µL 60%甲酸。将反应物在室温下再温育60分钟。荧光产物2，7-萘啶使用Tecan阅读器在375 nm处激发，430 nm处发射进行测量。IC50值是通过使用GraphPad Prism软件使用四参数S形剂量反应拟合抑制曲线（抑制百分比与抑制剂浓度）来确定的。

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使用LC-MS/MS检测的人NNMT酶测定[1]
使用适合目的的LC-MS/MS方法测量人NNMT反应中形成的MNA。将不同浓度的抑制剂与5ng/孔的人NNMT酶在室温下预孵育30分钟。通过分别加入7µM和20µM的SAM和烟酰胺混合物引发反应，并在37°C下孵育60分钟。最终的测定反应混合物包含100mM Tris-Hcl pH 7.5的缓冲液、0.04%BSA、2mM二硫苏糖醇和1%DMSO。向孔中加入100µL含内标d4-MNA（20ng/mL）的乙腈，并在室温下孵育10分钟。向孔中加入70µL高压灭菌水并轻轻混合。将该板在室温下以5000g离心10分钟。将150µL上清液转移到96孔板中，并通过LC-MS/MS进行分析。通过使用四参数S形剂量反应图垫棱镜拟合抑制曲线（抑制百分比与抑制剂浓度）来确定IC50值。

基于细胞的U2OS检测[1]
人骨骨肉瘤（U2OS）细胞系购自ATCC，并在含有10%热灭活胎牛血清和penstrep（过滤器灭菌）的DMEM F-12生长培养基中在37°C和5%CO2下维持。计数细胞，将每孔10K细胞接种到96孔细胞培养板中，然后在37°C、5%CO2和95%湿度下孵育24小时。将细胞培养基替换为100µl不同浓度的培养基/抑制剂混合物，并在37°C、5%CO2和95%湿度下孵育24小时。移除培养基/化合物混合物并洗涤两次，然后向孔中加入100µL含乙腈的内标d4-MNA（20ng/mL）。将该板在5000g下离心10分钟。将150µL上清液转移到96孔板（Costar 3364）中，并通过LC-MS/MS进行分析。通过使用四参数S形剂量反应图垫棱镜拟合抑制曲线（抑制百分比与抑制剂浓度）来确定IC50值。

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基于细胞的3T3L1检测[1]
3T3-L1是一种来源于小鼠3T3细胞的细胞系，从ATCC获得，并在含有10%热灭活胎牛血清和5%CO2的DMEM高糖生长培养基中在37°C下保持。计数细胞，将每孔5K细胞接种到96孔细胞培养板中，在37°C、5%CO2、95%湿度下孵育，直至达到100%融合。融合24小时后，用含有（500μM，IBMX+1μM DEXA+1μg/ml胰岛素）的分化诱导培养基替换培养基，并隔日更换培养基。细胞分化长达14天：三天后，用补充了10%FBS和1µg/ml胰岛素的DMEM代替诱导培养基。从第5天开始，细胞在常规生长培养基中培养。
分化后，细胞与化合物在37°C、5%CO2和95%湿度下孵育24小时。用DPBS洗涤细胞，并将100µl含内标d4 MNA（终浓度20ng/mL）的乙腈加入孔中。使用100%乙腈从细胞中提取MNA。将平板在室温下孵育20分钟，加入100µL高压水并轻轻混合。将该板在5000g下离心10分钟。将上清液转移到96孔板（Costar 3364）中，并提交LCMS/MS。通过使用四参数S形剂量反应图垫棱柱拟合抑制曲线（抑制百分比与抑制剂浓度）来确定IC50值。

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细胞毒性试验[1]
人肝癌癌症（HepG2）细胞系从ATCC获得，并在含有10%HI FBS和1%Pen-Strep（过滤灭菌）的DMEM Glut Max生长培养基中，在37°C、含5%CO2的培养箱中维持。当细胞在细胞培养瓶中80%融合时，用0.25%胰蛋白酶分离细胞。计数细胞，将每孔50 K细胞接种到96孔不透明壁多孔板中，然后在37°C、5%CO2、95%湿度下孵育过夜。将细胞培养基替换为100µL培养基/抑制剂混合物（含0.5%DMSO）和对照，并在37°C、5%CO2、95%湿度下孵育48或72小时。将最大对照样品（与0.5%DMSO完全反应）和最小对照样品（和已知抑制剂完全反应）Cell Titer Glo®试剂以1:1的比例添加到所有含有培养基的孔中（例如，将100µL试剂添加到96孔板的100µL含有细胞的培养基中）。将内容物在轨道振荡器上混合2分钟以诱导细胞裂解。然后将该板在室温下孵育10分钟以稳定发光信号。发光记录在Victor或Top Count发光计数器中。计算DMSO对照的细胞存活率。

Animal/Disease Models: High-fat diet (HFD)-induced obese mice [1]
Doses: 50 mg/kg
Route of Administration: Oral route for 4 weeks; the blocking bioavailability was found to be approximately 40% [1]. po (oral gavage) administration twice (two times) daily for four weeks
Experimental Results: Demonstrated significant weight loss (%) and resulted in a significant reduction in postprandial blood glucose by the oral route on day 21. On day 28, there was a statistically significant improvement in oral glucose tolerance, which was normalized by po (oral gavage).

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Animal/Disease Models: C57BL/6 mice[1]
Doses: 1 mg/kg (intravenous (iv) (iv)administration); 10 mg/kg (po (oral gavage)) (pharmacokinetic/PK/PK study)
Route of Administration: intravenous (iv) (iv)administration and po (oral gavage) ; 4 hour
Experimental Results: resulting in a low plasma clearance of 21 mL/min·kg, a steady-state volume of distribution of 0.7 L/kg, and a very short plasma half-life of 0.5 hrs (hrs (hours)) after intravenous (iv) (iv)injection. The results demonstrated that the Cmax was 3568 ng/mL, and the Tmax value was 0.5 hrs (hrs (hours)), indicating ra

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Efficacy studies[1]
Lean control + Vehicle and HFD + Vehicle Control groups (G1 and G2) mice were administered with vehicle. Dose formulations of HFD + JBSNF-000088, 50 mg kg−1, po, bid were administered at dose volume of 5 mL kg−1 body weight to G3 mice. Similar pattern was followed for ob/ob mice and db/db mice studies. The dose volume for individual animals was calculated based on the most recently recorded body weight during the study period. Throughout the study period, all animals were observed for mortality/morbidity. Cage side observations of animals for visible clinical signs, was carried out once daily throughout the study period. Individual animal body weights were recorded twice weekly during the study period.

The ob/ob mice were 12 weeks of age at the start of the study and db/db mice were 8 weeks of age at the start of the study. DIO mice were 20 weeks of age at the start of the study. In each study, non-diabetic lean mice comprised the control group which received vehicle (0.5% w/v HEC and 0.5% v/v Tween 80) and obese or diabetic mice were randomly assigned to two groups based on body weight and unfasted glucose which received either vehicle (0.5% w/v HEC and 0.5% v/v Tween 80) or JBSNF-000088, 50 mg kg−1, po, bid for 30 days. Individual animal body weights, food and water consumption were recorded twice weekly during the study period. Each group consisted of 10 animals in DIO, ob/ob and db/db efficacy studies. Animals were housed as n = 5 per cage and individual animal body weight, food and water consumption were recorded twice weekly for the duration of the study. Food consumption was expressed as cumulative energy intake. Fed blood glucose and insulin were measured on day 7, 14 and day 21 post treatment.

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OGTT[1]
Effect of JBSNF-000088 on glucose tolerance was assessed in an oral glucose tolerance test (OGTT) on day 28 of treatment, in DIO mice, ob/ob mice and on day 26 of treatment for db/db mice. Animals from DIO and ob/ob study were fasted for 4 h followed by an oral administration of glucose (2 g kg−1) while animals from db/db study were fasted for 16 h followed by an oral administration of glucose (1 g kg−1). One hour prior to glucose administration, mice were dosed orally with vehicle or JBSNF-000088, 50 mg kg−1.

Blood glucose measurements from tail snips were performed at −60 (prior to drug administration), 0 (prior to glucose administration), and 15, 30, 60, 120 and 180 minutes after glucose administration. Blood for plasma insulin measurement was collected at 0 and 15 minutes. HOMA-IR was calculated according to the formula; [HOMA-IR = (Fasting plasma insulin, ngml−1 × Fasting Blood Glucose, mmol l−1) / 22.5]. Animals were re-fed after the last time point of blood glucose and dosing was continued until termination on day 30.

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Study termination[1]
On day 30 of treatment, animals were fasted for 4 h and sacrificed by CO2 asphyxiation. One h prior to sacrifice, mice were dosed orally with vehicle or JBSNF-000088, 50 mg kg−1. Blood and tissue samples (liver, subcutaneous fat, renal fat, epididymal fat and mesentery fat) were collected from each animal. Plasma and tissue samples were stored at −80 °C until analysis.

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DIO studies using WT and NNMT KO animals[1]
NNMT fl/fl mice on a C57BL/6 background were generated using VelociGene® technology31. A loxP locus was placed 1.6 kb upstream of the transcription starting site encompassing the gene promoter. A loxP-Frt-Hyg-Frt cassette was inserted 212 bp downstream of NNMT exon 1. The floxed coordinates were chr9:48,412,881–48,414,698. NNMT fl/fl mice were crossed with ZP3-cre mice (C57BL/6, Jackson Laboratories) to generate whole-body NNMT knockout mice. The DIO mouse study was conducted in accordance to the German Animal Protection Law, as well as according to international animal welfare legislation and rules. Female wild-type and NNMT knockout animals were put on high-fat diet (Ssniff HFD adjusted TD.97366) for 18 weeks and then treated with either
(50 mg/kg bid by oral gavage) or vehicle for four weeks (n = 9–10 per group). Throughout the study, Mice were housed in an environmentally controlled room at 23 °C on a 12 h:12 h light dark cycle (light on at 06:00 AM), and food and water was offered ad libitum. An oral glucose tolerance test was performed on day 25 of treatment: Animals were fasted overnight. 60 minutes before an oral glucose bolus (2 g kg−1), JBSNF-000088 (50 mg kg−1) or vehicle (0.5% HEC + 0.5% Tween80) was administered by oral gavage. For blood glucose analysis, blood was collected from the tail of conscious mice at time points 15, 30, 60 and 120 minutes after the glucose bolus.

[1]. A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders. Sci Rep. 2018 Feb 26;8(1):3660.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.[1]

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Our attempts to determine the binding mode of JBSNF-000088 using mouse and human NNMT proteins by X-ray crystallography led to the finding that JBSNF-000088 is methylated and the methylated form of JBSNF-000088 is bound to the active substrate binding site in both proteins with the co-factor SAM turning into the demethylated form (SAH). This supports the hypothesis that JBSNF-000088 may act as a substrate analog, competing with the natural substrate NA and inhibiting the binding of NA to the active site of the enzyme. Once bound to the active site, transfer of methyl group from SAM can lead to the formation of N-methylated JBSNF-000088. Interestingly, the N-methylated product was also found in the circulating plasma of animals dosed with the un-methylated small molecule (JBSNF-000088), though only to a small extent. The latter may be explained by JBSNF-000088 being a potent binder but poor substrate for NNMT with resulting slow turnover. However, in the NNMT co-crystal, due to the high enzyme concentration, formation of the N-methylated compound was clearly detectable. The N-methylated product of JBSNF-000088 itself is a poor inhibitor of NNMT, with an IC50 value of > 30 µM (20% inhibition at 30 µM). Therefore, JBSNF-000088 may act as a competitive substrate analog that is very slowly converted to its N-methylated product. To our knowledge, this is the first proof-of-concept study using a small molecule modulator of NNMT in animal models of metabolic disease to demonstrate pharmacological benefits. Our study opens up the possibility of developing small molecule modulators of NNMT to test in patients with metabolic disorders.[1]

C7H8N2O2

152.15

152.059

C, 55.26; H, 5.30; N, 18.41; O, 21.03

7150-23-4

7150-23-4

250810

White to light yellow solid powder

1.213g/cm3

301.6ºC at 760mmHg

136.2ºC

1.55

0.889

65.21

1

3

2

11

149

0

O(C([H])([H])[H])C1C([H])=C([H])C(C(N([H])[H])=O)=C([H])N=1

KXDSMFBEVSJYRF-UHFFFAOYSA-N

InChI=1S/C7H8N2O2/c1-11-6-3-2-5(4-9-6)7(8)10/h2-4H,1H3,(H2,8,10)

6-methoxypyridine-3-carboxamide

JBSNF-000088; 6-Methoxynicotinamide; 6-METHOXYNICOTINAMIDE; 7150-23-4; 6-methoxypyridine-3-carboxamide; CHEMBL4206972; 3-Pyridinecarboxamide,6-methoxy-; NSC70628; MFCD00229166; JBSNF 000088; JBSNF000088

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~30 mg/mL (~197.2 mM)
Ethanol: ~7 mg/mL (~46 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (13.67 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (13.67 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (13.67 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2.94 mg/mL (19.32 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。